2009 Rustbelt RNA Meeting
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Talk on Friday 01:25-01:40pm submitted by Arlie Rinaldi

Dissecting the interactions between the putative endonuclease Nob1 and pre-18S rRNA

Arlie Rinaldi (University of Michigan, Department of Chemistry), Allison Lamanna (University of Michigan, Department of Chemistry), Katrin Karbstein (University of Michigan, Department of Chemistry)

Abstract:
Ribosomes are the machinery responsible for catalyzing protein synthesis in all cells. In eukaryotes, the two ribosomal subunits contain four ribosomal RNA (rRNA) molecules and 78 ribosomal proteins [1]. Eukaryotic ribosome assembly is a very complex process, requiring the action of more than 250 transiently associated protein and RNA accessory factors. These factors are required for precise folding and processing of the rRNAs, as well as for correct incorporation of the ribosomal proteins. While the identities of the necessary assembly factors are known, many of their functions have yet been elucidated. The Saccharomyces cerevisiae protein Nob1 is required for cell viability. Depletion of Nob1 results in a defect in 40S, or small subunit processing. Specifically, it leads to an accumulation of 20S rRNA, the direct precursor to mature 18S rRNA, which is the sole rRNA component of the small subunit [2]. Nob1 contains a N-terminal PIN domain, a ~120 amino acid domain characterized by four conserved acidic amino acids that serve to chelate a metal ion necessary for nuclease activity. Additionally, Nob1 contains a zinc ribbon domain. Nob1 has been shown to copurify with cytoplasmic pre-40S ribosomes [3] along with six other trans-acting factors [4]. Since Nob1 is the only factor bound to pre-40S ribosomes that contains a nuclease domain, it is believed to be the endonuclease responsible for the cleavage event that produces mature 18S rRNA. Here, we show that mutation of basic residues within the protein’s zinc ribbon domain, as well as truncation of its C-terminus results in decreased rRNA binding affinity in vitro accompanied with an increased growth rate in vivo.

References:
1. Fromont-Racine M, et al. (2003) Gene 313, 17-42.
2. Fatica A, et al. (2003) Mol Cell Biol 23, 1798-1807.
3. Schäfer T, et al. (2006) Nature 441, 651-655.
4. Schäfer T, et al. (2003) EMBO J 22, 1370-1380.

Keywords: ribosome assembly, endonuclease